Substituted Fused Pyrrolo-Diazepinones and Uses Thereof

ABSTRACT

Disclosed are compounds, pharmaceutical compositions, and methods of treatment. The disclosed compounds are based on fused 1,4-diazepine and pyrrolidinedione scaffolds. The compounds may be utilized in pharmaceutical compositions and methods for treating diseases and disorders associated with cell proliferation such as cancer and may have a formula illustrated as follows:

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62/639,770, the content ofwhich is incorporated herein by reference in its entirety.

BACKGROUND

The field of the invention relates to fused pyrrolo-diazepinones. Inparticular, the field of the invention relates to substituted fusedpyrrolo-diazepinones and uses thereof for treating diseases anddisorders such as cell proliferative diseases and disorders such ascancers.

Over 200,000 people a year in the U.S. are diagnosed with lung cancereach year. It is by far the leading cause of cancer death among both menand women. The lung cancer five-year survival rate (17.7 percent) islower than many other leading cancer sites, such as the colon (64.4percent), breast (89.7 percent) and prostate (98.9 percent). Thefive-year survival rate for lung cancer is 55 percent for cases detectedwhen the disease is still localized (within the lungs). However, only 16percent of lung cancer cases are diagnosed at an early stage. Fordistant tumors (spread to other organs) the five-year survival rate isonly 4 percent. More than half of people with lung cancer die within oneyear of being diagnosed.

Existing small molecule agents for lung cancer are typically alkylatingagents of microtubule stabilizing agents, each of which produces majorside effects. Development of new drugs for lung cancer and other cancersand persistent diseases will increasingly rely on the expansion ofaccessible chemical space to allow exploration of novel moleculartargets. Here we report the synthesis of a library of novel fusedheterobicyclic small molecules based on 1,4-diazepine and2,4-pyrrolidinedione scaffolds. Key chemical transformations included aMannich-type condensation and a chemoselective N-acylation reactions.Screening of these new compounds demonstrates that the new compoundshave anti-cancer activity which suggests translational potential of thisnovel chemical scaffold. Some compounds have particular efficacy againstlung cancer cells in vitro.

SUMMARY

Disclosed are compounds, pharmaceutical compositions, and methods oftreatment. The disclosed compounds are based on fused 1,4-diazepine andpyrrolidinedione scaffolds and may described as compounds having thefollowing core and substituted derivatives thereof:

The compounds may be utilized in pharmaceutical compositions and methodsfor treating diseases and disorders associated with cell proliferation.In particular, the compounds may be utilized in pharmaceuticalcompositions and methods for treating cancer.

The disclosed compounds may be formulated as pharmaceutical compositionscomprising one or more of the disclosed compounds, a tautomer thereof,or a pharmaceutical salt thereof, and a suitable pharmaceutical carrier.The disclosed compounds or pharmaceutical compositions comprising thedisclosed compounds may be administered to a subject in need thereof,for example, to treat a disease or disorder associated with cellproliferation such as cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Examples of pharmacologically relevant diazepine-containingcompounds.

FIG. 2. Representative natural products containing the tetramic acidscaffold.

FIG. 3. A459 cell viability after 3-day treatment with compoundNUCC-60855 at the indicated concentrations.

FIG. 4. A459 cell viability after 3-day treatment with compoundNUCC-60872 at the indicated concentrations.

FIG. 5. A459 cell viability after 3-day treatment with compoundNUCC-60854 or NU-77057 at the indicated concentrations.

FIG. 6. Cell viability data for compound NUCC-60855 against breastcancer cells and normal cells. Cells were treated for two (2) days.

FIG. 7. Percent viability of compounds (50 μM) against MDA-MB-468 breastcancer cells after treatment with the listed compounds.

FIG. 8. MDA-468 cell viability assay in the presence of compoundsNUCC-60855, NUCC-60872, NUCC-202782, and NUCC-202790.

FIG. 9. Additional derivative compound that can be synthesized by themethods disclosed herein.

FIG. 10. Additional derivative compound that can be synthesized by themethods disclosed herein where “R” indicates the point of attachment ofthe substituent to the fused core structure at the oxo group (topstructure) or at the diazepine nitrogen atom (bottom structure).

DETAILED DESCRIPTION

As used herein, unless otherwise specified or indicated by context, theterms “a”, “an”, and “the” mean “one or more.” For example, “a compound”or “a substituent” should be interpreted to mean “one or more compounds”and “one or more substituents,” respectively.

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus ≤10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

As used herein, a “subject” may be interchangeable with “patient” or“individual” and means an animal, which may be a human or non-humananimal, in need of treatment, for example, treatment by includeadministering a therapeutic amount of one or more compounds orpharmaceutical compositions as disclosed herein.

A “subject in need of treatment” may include a subject having a cellproliferative disease, disorder, or condition such as cancer. Cancersmay include, but are not limited to adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, and teratocarcinoma and particularly cancersof the adrenal gland, bladder, blood, bone, bone marrow, brain, breast,cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney,liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin,testis, thymus, and uterus.

Chemical Entities

New chemical entities and uses for chemical entities are disclosedherein. The chemical entities may be described using terminology knownin the art and further discussed below.

As used herein, an asterisk “*” or a plus sign “+” may be used todesignate the point of attachment for any radical group or substituentgroup.

The term “alkyl” as contemplated herein includes a straight-chain orbranched alkyl radical in all of its isomeric forms, such as a straightor branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to hereinas C₁-C₁₂ alkyl, C₁-C₁₀-alkyl, and C₁-C₆-alkyl, respectively.

The term “alkylene” refers to a diradical of an alkyl group (e.g.,—(CH₂)_(n)— where n is an integer such as an integer between 1 and 20).An exemplary alkylene group is —CH₂CH₂—.

The term “haloalkyl” refers to an alkyl group that is substituted withat least one halogen. For example, —CH₂F, —CHF₂, —CF₃, —CH₂CF₃, —CF₂CF₃,and the like.

The term “heteroalkyl” as used herein refers to an “alkyl” group inwhich at least one carbon atom has been replaced with a heteroatom(e.g., an O, N, or S atom). One type of heteroalkyl group is an “alkoxy”group.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C₂-C₁₂-alkenyl, C₂-C₁₀-alkenyl, and C₂-C₆-alkenyl,respectively.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms,referred to herein as C₂-C₁₂-alkynyl, C₂-C₁₀-alkynyl, and C₂-C₆-alkynyl,respectively.

The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic,or bridged cyclic (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8,or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derivedfrom a cycloalkane. Unless specified otherwise, cycloalkyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the cycloalkyl group is not substituted, i.e., itis unsubstituted.

The term “cycloalkylene” refers to a cycloalkyl group that isunsaturated at one or more ring bonds.

The term “partially unsaturated carbocyclyl” refers to a monovalentcyclic hydrocarbon that contains at least one double bond between ringatoms where at least one ring of the carbocyclyl is not aromatic. Thepartially unsaturated carbocyclyl may be characterized according to thenumber oring carbon atoms. For example, the partially unsaturatedcarbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, andaccordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 memberedpartially unsaturated carbocyclyl, respectively. The partiallyunsaturated carbocyclyl may be in the form of a monocyclic carbocycle,bicyclic carbocycle, tricyclic carbocycle, bridged carbocycle,spirocyclic carbocycle, or other carbocyclic ring system. Exemplarypartially unsaturated carbocyclyl groups include cycloalkenyl groups andbicyclic carbocyclyl groups that are partially unsaturated. Unlessspecified otherwise, partially unsaturated carbocyclyl groups areoptionally substituted at one or more ring positions with, for example,alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino,amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano,cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato,phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. Incertain embodiments, the partially unsaturated carbocyclyl is notsubstituted, i.e., it is unsubstituted.

The term “aryl” is art-recognized and refers to a carbocyclic aromaticgroup. Representative aryl groups include phenyl, naphthyl, anthracenyl,and the like. The term “aryl” includes polycyclic ring systems havingtwo or more carbocyclic rings in which two or more carbons are common totwo adjoining rings (the rings are “fused rings”) wherein at least oneof the rings is aromatic and, e.g., the other ring(s) may becycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unlessspecified otherwise, the aromatic ring may be substituted at one or morering positions with, for example, halogen, azide, alkyl, aralkyl,alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, carboxylic acid, —C(O)alkyl, —CO₂alkyl,carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide,ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties,—CF₃, —CN, or the like. In certain embodiments, the aromatic ring issubstituted at one or more ring positions with halogen, alkyl, hydroxyl,or alkoxyl. In certain other embodiments, the aromatic ring is notsubstituted, i.e., it is unsubstituted. In certain embodiments, the arylgroup is a 6-10 membered ring structure.

The terms “heterocyclyl” and “heterocyclic group” are art-recognized andrefer to saturated, partially unsaturated, or aromatic 3- to 10-memberedring structures, alternatively 3- to 7-membered rings, whose ringstructures include one to four heteroatoms, such as nitrogen, oxygen,and sulfur. The number of ring atoms in the heterocyclyl group can bespecified using 5 Cx-Cx nomenclature where x is an integer specifyingthe number of ring atoms. For example, a C3-C7 heterocyclyl group refersto a saturated or partially unsaturated 3- to 7-membered ring structurecontaining one to four heteroatoms, such as nitrogen, oxygen, andsulfur. The designation “C3-C7” indicates that the heterocyclic ringcontains a total of from 3 to 7 ring atoms, inclusive of any heteroatomsthat occupy a ring atom position.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines (e.g., mono-substituted amines ordi-substituted amines), wherein substituents may include, for example,alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.

The terms “alkoxy” or “alkoxyl” are art-recognized and refer to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxy groups include methoxy, ethoxy, tert-butoxy andthe like.

An “ether” is two hydrocarbons covalently linked by an oxygen.

Accordingly, the substituent of an alkyl that renders that alkyl anether is or resembles an alkoxyl, such as may be represented by one of—O-alkyl, —O-alkenyl, —O-alkynyl, and the like.

The term “carbonyl” as used herein refers to the radical —C(O)—.

The term “oxo” refers to a divalent oxygen atom —O—.

The term “carboxamido” as used herein refers to the radical —C(O)NRR′,where R and R′ may be the same or different. R and R, for example, maybe independently alkyl, aryl, arylalkyl, cycloalkyl, formyl, haloalkyl,heteroaryl, or heterocyclyl.

The term “carboxy” as used herein refers to the radical —COOH or itscorresponding salts, e.g. —COONa, etc.

The term “amide” or “amido” or “amidyl” as used herein refers to aradical of the form —R′C(O)N(R²)—, —R′C(O)N(R²)R³—, —C(O)NR²R³, or—C(O)NH₂, wherein R′, R² and R³, for example, are each independentlyalkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl,heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.

The term “amino protecting group” is known in the art and may include,but is not limited to t-butyl carbamate (BOC), p-methoxybenzyl (PMB),9-fluorenylmethyl carbamate (FMOC), benzyl carbamate (Cbz), acetamide(Ac), trifluoroacetamide, phthalimide, benzylamine (Bn),triphenylmethylamine (Tr), benzylideneamine, and p-toluenesulfonamide(Ts).

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” or “+” or “−” depending on the configuration ofsubstituents around the stereogenic carbon atom and or the opticalrotation observed. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers includeenantiomers and diastereomers. Mixtures of enantiomers or diastereomersmay be designated (±)” in nomenclature, but the skilled artisan willrecognize that a structure may denote a chiral center implicitly. It isunderstood that graphical depictions of chemical structures, e.g.,generic chemical structures, encompass all stereoisomeric forms of thespecified compounds, unless indicated otherwise. Also contemplatedherein are compositions comprising, consisting essentially of, orconsisting of an enantiopure compound, which composition may comprise,consist essential of, or consist of at least about 50%, 60%, 70%, 80%,90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a givencompound (e.g., at least about 99% of an R enantiomer of a givencompound). The compounds may have an undefined double stereo bond whosesubstituents may be present in either of the syn-conformation or theanti-conformation (or alternatively in the E-conformation or theZ-conformation).

Substituted Fused Pyrrolo-Diazepinones and Uses Thereof

Disclosed are compounds, pharmaceutical compositions, and method oftreatment. The disclosed compounds are based on fused 1,4-diazepine andpyrrolidinedione scaffolds and may described as compounds having thefollowing core or substituted derivatives thereof:

The compounds may be utilized in pharmaceutical compositions and methodsfor treating diseases and disorders associated with cell proliferation.In particular, the compounds may be utilized in pharmaceuticalcompositions and methods for treating cancer.

The disclosed compounds may be formulated as pharmaceutical compositionscomprising one or more of the disclosed compounds, a tautomer thereof,or a pharmaceutical salt thereof, and a suitable pharmaceutical carrier.The disclosed compounds or pharmaceutical compositions comprising thedisclosed compounds may be administered to a subject in need thereof,for example, to treat a disease or disorder associated with cellproliferation such as cancer.

The compounds disclosed herein may exist in one or more tautomeric formsas known in the art. The disclosed compounds therefore encompasstautomeric derivatives as would be known in the art. The compoundsdisclosed herein may have several chiral centers, and stereoisomers,epimers, and enantiomers are contemplated. The compounds may beoptically pure with respect to one or more chiral centers (e.g., some orall of the chiral centers may be completely in the S configuration; someor all of the chiral centers may be completely in the R configuration;etc.). Additionally or alternatively, one or more of the chiral centersmay be present as a mixture of configurations (e.g., a racemic oranother mixture of the R configuration and the S configuration).Compositions comprising substantially purified stereoisomers, epimers,or enantiomers, or analogs or derivatives thereof are contemplatedherein (e.g., a composition comprising at least about 90%, 95%, or 99%pure stereoisomer, epimer, or enantiomer.) As used herein, formulaewhich do not specify the orientation at one or more chiral centers aremeant to encompass all orientations and mixtures thereof.

Pharmaceutical Compositions

The compounds employed in the compositions and methods disclosed hereinmay be administered as pharmaceutical compositions and, therefore,pharmaceutical compositions incorporating the compounds are consideredto be embodiments of the compositions disclosed herein. Suchcompositions may take any physical form which is pharmaceuticallyacceptable; illustratively, they can be orally administeredpharmaceutical compositions. Such pharmaceutical compositions contain aneffective amount of a disclosed compound, which effective amount isrelated to the daily dose of the compound to be administered. Eachdosage unit may contain the daily dose of a given compound or eachdosage unit may contain a fraction of the daily dose, such as one-halfor one-third of the dose. The amount of each compound to be contained ineach dosage unit can depend, in part, on the identity of the particularcompound chosen for the therapy and other factors, such as theindication for which it is given. The pharmaceutical compositionsdisclosed herein may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing well known procedures.

The compounds for use according to the methods of disclosed herein maybe administered as a single compound or a combination of compounds. Forexample, a compound that treats cancer may be administered as a singlecompound or in combination with another compound that treats cancer orthat has a different pharmacological activity.

As indicated above, pharmaceutically acceptable salts of the compoundsare contemplated and also may be utilized in the disclosed methods. Theterm “pharmaceutically acceptable salt” as used herein, refers to saltsof the compounds which are substantially non-toxic to living organisms.Typical pharmaceutically acceptable salts include those salts preparedby reaction of the compounds as disclosed herein with a pharmaceuticallyacceptable mineral or organic acid or an organic or inorganic base. Suchsalts are known as acid addition and base addition salts. It will beappreciated by the skilled reader that most or all of the compounds asdisclosed herein are capable of forming salts and that the salt forms ofpharmaceuticals are commonly used, often because they are more readilycrystallized and purified than are the free acids or bases.

Acids commonly employed to form acid addition salts may includeinorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodicacid, sulfuric acid, phosphoric acid, and the like, and organic acidssuch as p-toluenesulfonic, methanesulfonic acid, oxalic acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of suitablepharmaceutically acceptable salts may include the sulfate, pyrosulfate,bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate, formate,hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate,propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate,maleat-, butyne-.1,4-dioate, hexyne-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate,phthalate, xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, alpha-hydroxybutyrate, glycolate,tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like.

Base addition salts include those derived from inorganic bases, such asammonium or alkali or alkaline earth metal hydroxides, carbonates,bicarbonates, and the like. Bases useful in preparing such salts includesodium hydroxide, potassium hydroxide, ammonium hydroxide, potassiumcarbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate, and the like.

The particular counter-ion forming a part of any salt of a compounddisclosed herein is may not be critical to the activity of the compound,so long as the salt as a whole is pharmacologically acceptable and aslong as the counterion does not contribute undesired qualities to thesalt as a whole. Undesired qualities may include undesirably solubilityor toxicity.

Pharmaceutically acceptable esters and amides of the compounds can alsobe employed in the compositions and methods disclosed herein. Examplesof suitable esters include alkyl, aryl, and aralkyl esters, such asmethyl esters, ethyl esters, propyl esters, dodecyl esters, benzylesters, and the like. Examples of suitable amides include unsubstitutedamides, monosubstituted amides, and disubstituted amides, such as methylamide, dimethyl amide, methyl ethyl amide, and the like.

In addition, the methods disclosed herein may be practiced using solvateforms of the compounds disclosed herein or salts, esters, and/or amides,thereof. Solvate forms may include ethanol solvates, hydrates, and thelike.

As used herein, the terms “treating” or “to treat” each mean toalleviate symptoms, eliminate the causation of resultant symptoms eitheron a temporary or permanent basis, and/or to prevent or slow theappearance or to reverse the progression or severity of resultantsymptoms of the named disease or disorder. As such, the methodsdisclosed herein encompass both therapeutic and prophylacticadministration.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of subjects in need of suchtreatment. An effective amount of a drug that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

An effective amount can be readily determined by the attendingdiagnostician, as one skilled in the art, by the use of known techniquesand by observing results obtained under analogous circumstances. Indetermining the effective amount or dose of compound administered, anumber of factors can be considered by the attending diagnostician, suchas: the species of the subject; its size, age, and general health; thedegree of involvement or the severity of the disease or disorderinvolved; the response of the individual subject; the particularcompound administered; the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the use of concomitant medication; and other relevantcircumstances.

A typical daily dose may contain from about 0.01 mg/kg to about 100mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about0.1 mg/kg to about 25 mg/kg) of each compound used in the present methodof treatment.

Compositions can be formulated in a unit dosage form, each dosagecontaining from about 1 to about 500 mg of each compound individually orin a single unit dosage form, such as from about 5 to about 300 mg, fromabout 10 to about 100 mg, and/or about 25 mg. The term “unit dosageform” refers to a physically discrete unit suitable as unitary dosagesfor a patient, each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier, diluent, orexcipient.

Oral administration is an illustrative route of administering thecompounds employed in the compositions and methods disclosed herein.Other illustrative routes of administration include transdermal,percutaneous, intravenous, intramuscular, intranasal, buccal,intrathecal, intracerebral, or intrarectal routes. The route ofadministration may be varied in any way, limited by the physicalproperties of the compounds being employed and the convenience of thesubject and the caregiver.

As one skilled in the art will appreciate, suitable formulations includethose that are suitable for more than one route of administration. Forexample, the formulation can be one that is suitable for bothintrathecal and intracerebral administration. Alternatively, suitableformulations include those that are suitable for only one route ofadministration as well as those that are suitable for one or more routesof administration, but not suitable for one or more other routes ofadministration. For example, the formulation can be one that is suitablefor oral, transdermal, percutaneous, intravenous, intramuscular,intranasal, buccal, and/or intrathecal administration but not suitablefor intracerebral administration.

The inert ingredients and manner of formulation of the pharmaceuticalcompositions are conventional. The usual methods of formulation used inpharmaceutical science may be used here. All of the usual types ofcompositions may be used, including tablets, chewable tablets, capsules,solutions, parenteral solutions, intranasal sprays or powders, troches,suppositories, transdermal patches, and suspensions. In general,compositions contain from about 0.5% to about 50% of the compound intotal, depending on the desired doses and the type of composition to beused. The amount of the compound, however, is best defined as the“effective amount”, that is, the amount of the compound which providesthe desired dose to the patient in need of such treatment. The activityof the compounds employed in the compositions and methods disclosedherein are not believed to depend greatly on the nature of thecomposition, and, therefore, the compositions can be chosen andformulated primarily or solely for convenience and economy.

Capsules are prepared by mixing the compound with a suitable diluent andfilling the proper amount of the mixture in capsules. The usual diluentsinclude inert powdered substances (such as starches), powdered cellulose(especially crystalline and microcrystalline cellulose), sugars (such asfructose, mannitol and sucrose), grain flours, and similar ediblepowders.

Tablets are prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants, and disintegrators (in addition to the compounds).Typical diluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts (such assodium chloride), and powdered sugar. Powdered cellulose derivatives canalso be used. Typical tablet binders include substances such as starch,gelatin, and sugars (e.g., lactose, fructose, glucose, and the like).Natural and synthetic gums can also be used, including acacia,alginates, methylcellulose, polyvinylpyrrolidine, and the like.Polyethylene glycol, ethylcellulose, and waxes can also serve asbinders.

Tablets can be coated with sugar, e.g., as a flavor enhancer andsealant. The compounds also may be formulated as chewable tablets, byusing large amounts of pleasant-tasting substances, such as mannitol, inthe formulation. Instantly dissolving tablet-like formulations can alsobe employed, for example, to assure that the patient consumes the dosageform and to avoid the difficulty that some patients experience inswallowing solid objects.

A lubricant can be used in the tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant can be chosen fromsuch slippery solids as talc, magnesium and calcium stearate, stearicacid, and hydrogenated vegetable oils.

Tablets can also contain disintegrators. Disintegrators are substancesthat swell when wetted to break up the tablet and release the compound.They include starches, clays, celluloses, algins, and gums. As furtherillustration, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp, sodium lauryl sulfate, andcarboxymethylcellulose can be used.

Compositions can be formulated as enteric formulations, for example, toprotect the active ingredient from the strongly acid contents of thestomach. Such formulations can be created by coating a solid dosage formwith a film of a polymer which is insoluble in acid environments andsoluble in basic environments. Illustrative films include celluloseacetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and hydroxypropyl methylcellulose acetatesuccinate.

When it is desired to administer the compound as a suppository,conventional bases can be used. Illustratively, cocoa butter is atraditional suppository base. The cocoa butter can be modified byaddition of waxes to raise its melting point slightly. Water-misciblesuppository bases, such as polyethylene glycols of various molecularweights, can also be used in suppository formulations.

Transdermal patches can also be used to deliver the compounds.Transdermal patches can include a resinous composition in which thecompound will dissolve or partially dissolve; and a film which protectsthe composition and which holds the resinous composition in contact withthe skin. Other, more complicated patch compositions can also be used,such as those having a membrane pierced with a plurality of poresthrough which the drugs are pumped by osmotic action.

As one skilled in the art will also appreciate, the formulation can beprepared with materials (e.g., actives excipients, carriers (such ascyclodextrins), diluents, etc.) having properties (e.g., purity) thatrender the formulation suitable for administration to humans.Alternatively, the formulation can be prepared with materials havingpurity and/or other properties that render the formulation suitable foradministration to non-human subjects, but not suitable foradministration to humans.

The following list of formulations is illustrative. These illustrativeformulations may be suitable for preparing pharmaceutical compositionsthat include the disclosed compounds as “active ingredients.” Thefollowing list of formulations is illustrative and should not beinterpreted as limiting the present disclosure or claims in any way:

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity (mg/capsule) Active Ingredient 250 Starch, dried 200 Magnesiumstearate 10 Total 460 mg

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

Formulation 2

Quantity (mg/tablet) Active Ingredient 250 Cellulose, microcrystalline400 Silicon dioxide, fumed 10 Stearic acid 5 Total 665 mg

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

Weight % Active Ingredient 0.25 Ethanol 29.75 Propellant 22(chlorodifluoromethane) 70.00 Total 100.00

The active compound is mixed with ethanol and the mixture added to aportion of the Propellant 22, cooled to ⁻30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets each containing 60 mg of active ingredient are made as follows:

Active Ingredient 60 mg Starch 45 mg Microcrystalline cellulose 35 mgPolyvinylpyrrolidone 4 mg Sodium carboxymethyl starch 4.5 mg Magnesiumstearate 0.5 mg Talc 1 mg Total 150 mg

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation 5

Capsules, each containing 80 mg medicament, are made as follows:

Active Ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mgMagnesium stearate 2 mg Total 200 mg

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 sieve, and filled into hard gelatincapsules in 200 mg quantities.

Formulation 6

Suppositories each containing 225 mg of active ingredient may be made asfollows:

Active Ingredient 225 mg Saturated fatty acid glycerides 2,000 mg Total2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions each containing 50 mg of medicament per 5 ml dose are madeas follows:

Active Ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25ml Benzoic acid solution 0.10 ml Flavor q.v. Color q.v. Purified waterto total 5 ml

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl, cellulose and syrup to form a smooth paste.The benzoic acid solution, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

An intravenous formulation containing 100 mg of medicament per 5 ml dosecan be prepared as follows:

Active Ingredient 100 mg Mannitol 100 mg 5N Sodium hydroxide 200 mlPurified water to total 5 n

ILLUSTRATIVE EMBODIMENTS

The following embodiments are illustrative and should not be interpretedto limit the scope of the claimed subject matter.

Embodiment 1

A compound or a salt thereof having a formula:

wherein:

-   R¹ and R² are H or alkyl;-   R³ is H, alkyl, alkenyl (e.g. isopentenyl), or benzyl;-   R⁴ is H, alkyl (e.g., methyl or isobutyl), benzyl, or a carbocycle    (e.g., a 3-, 4-, 5-, or 6-membered carbocycle such as cycloalkyl or    phenyl), a heterocycle (e.g., a 3-, 4-, 5-, or 6-membered    heterocycle such as piperidinyl, pyridinyl, or thiophenyl), two    fused carbocycles (e.g., two fused 3-, 4-, 5-, or 6-membered    carbocycles such as naphthalene), two fused heterocycles (e.g., two    fused 3-, 4-, 5-, or 6-membered heterocycles such as purine), or a    fused carbocycle and a fused heterocycle (e.g., a fused 3-, 4-, 5-,    or 6-membered carbocycle and a fused 3-, 4-, 5-, or 6-membered    heterocycle such as 1,3-benzodioxolyl or 1,4-benzodioxanyl), which    carbocycle and heterocycle are saturated or unsaturated at one or    more bonds and optionally are substituted at one or more positions    with halo (e.g., 3,4-dichlorophenyl), alkyl, haloalkyl, methoxy    (e.g. 4-methoxyphenyl), or carboxyl (e.g., 4-carboxyphenyl), or    carboxy;-   R^(4′) is H or alkyl;-   R⁵ is H, alkyl, aryl such as phenyl, alkylaryl such as benzyl,    sulfonyl, and R⁵ optionally is substituted at one or more positions    with alkyl, halo, haloalkyl, alkoxy, cyano, amino or    alkyl-substituted amino, or R⁵ has a formula

-    where R¹⁰ is H, alkyl, phenylamino, benzyl, or R¹⁰ is a carbocycle    (e.g., a 3-, 4-, 5-, or 6-membered carbocycle such as cycloalkyl or    phenyl), a heterocycle (e.g., a 3-, 4-, 5-, or 6-membered    heterocycle such as piperidinyl, pyridinyl, or thiophenyl), two    fused carbocycles (e.g., two fused 3-, 4-, 5-, or 6-membered    carbocycles such as naphthalene), two fused heterocycles (e.g., two    fused 3-, 4-, 5-, or 6-membered heterocycles such as purine), or a    fused carbocycle and a fused heterocycle (e.g., a fused 3-, 4-, 5-,    or 6-membered carbocycle and a fused 3-, 4-, 5-, or 6-membered    heterocycle such as 1,3-benzodioxolyl or 1,4-benzodioxanyl), which    carbocycle and heterocycle are saturated or unsaturated at one or    more bonds and R¹⁰ optionally is substituted at one or more    positions with halo, alkyl, alkoxy (e.g., where R¹⁰ is    4-methoxyphenyl), amino or substituted amino which optionally is    alkylamino or dialkylamino (e.g., where R¹⁰ is    4-(N,N-dimethylamino)phenyl), haloalkyl (e.g., where R¹⁰ is    4-trifluormethylphenyl) or carboxy; or R¹⁰ has a formula

-    wherein R¹³ is H or alkyl, R¹⁴ is H or alkyl or R¹³ and R¹⁴    together form cycloalkyl, a R¹⁵ is benzyl or oxybenzyl, or a    carbocycle (e.g., a 3-, 4-, 5-, or 6-membered carbocycle such as    cycloalkyl or phenyl), a heterocycle (e.g., a 3-, 4-, 5-, or    6-membered heterocycle such as piperidinyl, pyridinyl, or    thiophenyl), two fused carbocycles (e.g., two fused 3-, 4-, 5-, or    6-membered carbocycles such as naphthalene), two fused heterocycles    (e.g., two fused 3-, 4-, 5-, or 6-membered heterocycles such as    purine), or a fused carbocycle and a fused heterocycle (e.g., a    fused 3-, 4-, 5-, or 6-membered carbocycle and a fused 3-, 4-, 5-,    or 6-membered heterocycle such as 1,3-benzodioxolyl or    1,4-benzodioxanyl), which carbocycle and heterocycle are saturated    or unsaturated at one or more bonds and R¹⁵ optionally is    substituted at one or more positions with halo, alkyl, haloalkyl,    alkoxy, amino or substituted amino which optionally is alkylamino or    dialkylamino, or carboxy; and-   R⁶ and R⁷ are H, or R⁶ and R⁷ together form phenyl fused to the    1.4-diazepine core.

Embodiment 2

The compound of embodiment 1, wherein R¹ and R² are methyl.

Embodiment 3

The compound of embodiment 1 or 2, wherein R³ is methyl.

Embodiment 4

The compound of any of the foregoing embodiments, wherein R⁴ is selectedfrom methyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, furanyl (e.g., furan-2-yl), pyridinyl (e.g., pyridin-2-yl,pyridin-3-yl or pyridin-4-yl), phenyl or fluorophenyl (e.g.,4-flurophenyl) or chlorophenyl (e.g., 4-chlorophenyl or3,4-dichlorophenyl) or carboxyphenyl (e.g., 4-carboxyphenyl) ormethoxyphenyl (e.g., 4-methoxyphenyl), benzyl, thiophenyl (e.g.,thiophen-2-yl or thiophen-3-yl) or chlorothiophenyl (e.g.,5-chloro-thiophen-2-yl), tetrahydrofuranyl (e.g., tetrahydrofuran-2-ylor tetrahydrofuran-3-yl), thiazolyl (e.g., thiazol-2-yl),1,3-benzodioxolyl (e.g., 1,3-benzodioxol-5-yl), 1,4-benzodioxanyl (e.g.,1,4-benzodioxan-6-yl), and piperidinyl (e.g., 4-piperidinyl).

Embodiment 5

The compound of any of the foregoing embodiments, wherein R⁵ has aformula

and R¹⁰ is methyl, tetrabutyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, thiophenyl (e.g., thiophen-2-yl),4-(N,N-diakylamino)phenyl, 4-trifluoromethylphenyl, 4-methoxyphenyl,4-fluorophenyl, phenylamino, 1,3-benzodioxolyl (e.g.,1,3-benzodioxol-5-yl), 1,4-benzodioxanyl (e.g., 1,4-benzodioxan-6-yl),pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), amino oralkylamino (e.g., dialkylamino), phenyl, tetrahydropyranyl, (e.g.,tetrahydropyran-4-yl).

Embodiment 6

The compound of any of the foregoing embodiments, wherein R⁵ has aformula

R¹⁰ has a formula

R¹³ is H, and R¹⁴ is phenyl, oxybenzyl, tetrahydropyranyl, (e.g.,tetrahydropyran-4-yl), or 1,3-benzodioxolyl (e.g.,1,3-benzodioxol-5-yl).

Embodiment 7

The compound of any of the foregoing embodiments, wherein R⁴ is benzyland R⁵ is oxobenzyl.

Embodiment 8

A pharmaceutical composition comprising any effective amount of thecompound of any of the foregoing embodiments and a pharmaceuticalcarrier, excipient, or diluent.

Embodiment 9

A method for treating a disease or disorder in a subject in needthereof, the method comprising administering to the subject thepharmaceutical composition of embodiment 8.

Embodiment 10

The method of embodiment 9, wherein the disease or disorder is a cellproliferative disease or disorder.

Embodiment 11

The method of embodiment 9, wherein the disease or disorder is cancer,optionally wherein the cancer is selected from adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma and/or thecancer is selected from cancers of the adrenal gland, bladder, blood,bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, prostate, skin, testis, thymus, and uterus.

Embodiment 12

A compound of Table 2 in the Specification that accompanies theseembodiments.

Embodiment 13

A pharmaceutical composition comprising any effective amount of thecompound of embodiment 12 and a pharmaceutical carrier, excipient, ordiluent.

Embodiment 14

A method for treating a disease or disorder in a subject in needthereof, the method comprising administering to the subject thepharmaceutical composition of embodiment 13.

Embodiment 15

The method of embodiment 14, wherein the disease or disorder is a cellproliferative disease or disorder, optionally wherein the disease ordisorder is cancer, optionally wherein the cancer is selected fromadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma and/or the cancer is selected from cancers of theadrenal gland, bladder, blood, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis,thymus, and uterus.

Embodiment 16

A compound having a structure:

Embodiment 17

A compound having a structure:

wherein R¹ and R² are H or alkyl and R¹¹ and R¹² are H or an aminoprotecting group (e.g., t-butyl carbamate (BOC), p-methoxybenzyl (PMB),9-fluorenylmethyl carbamate (FMOC), benzyl carbamate (Cbz), acetamide(Ac), trifluoroacetamide, phthalimide, benzylamine (Bn),triphenylmethylamine (Tr), benzylideneamine, and p-toluenesulfonamide(Ts)).

Embodiment 18

The compound of embodiment 17, wherein R¹ and R² are methyl and R¹¹ andR¹² are H.

Embodiment 19

A compound having a structure:

wherein R¹ and R² are H or alkyl.

Embodiment 20

The compound of embodiment 19, wherein R¹ and R² are methyl.

Examples

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Synthesis of a Novel Fused Pyrrolodiazepine-Based Library withAnti-Cancer Activity

Reference is made to the manuscript entitled Malik et al., “Synthesis ofa Novel Fused Pyrrolodiazepine-Based Library with Anti-Cancer Activity,”Tetrahedron Letters, Volume 59, Issue 15, 11 Apr. 2018, Pages 1513-1516,the content of which is incorporated herein by reference in itsentirety.

Abstract

Development of drugs for new and persistent diseases will increasinglyrely on the expansion of accessible chemical space to allow explorationof novel molecular targets. Here we report the synthesis of a library ofnovel fused heterobicyclic small molecules based on the 1,4-diazepineand 2,4-pyrrolidinedione scaffolds. Key chemical transformationsincluded a Mannich-type condensation and chemoselective N-acylationreactions. Screening shows anti-cancer activity of several librarycompounds which suggests translational potential of this novel chemicalscaffold.

INTRODUCTION

There is a continuous need for the generation of small moleculelibraries to identify new lead compounds for drug discovery againstnovel targets: Incorporation of key scaffolds or pharmacophore-basedstructural features during the design of such small molecules aids instudying diverse biological targets. For instance, molecular frameworkscontaining the diazepine scaffold have been recognized to havesignificant medicinal relevance.² The benzodiazepines motif, forexample, is considered a ‘privileged structures’ with derivatives havinga broad range of biological activity such as against central nervoussystem disorders,³ anti-HIV,⁴⁻⁵ anti-malarial and anti-cancer⁶⁻⁷therapeutics. The benzodiazepine system is thought to have such wideutility in part because of its ability to mimic peptide β- and γ-turnmoieties.⁸

Synthetic agents and natural products containing the2,4-pyrrolidinedione ring system or tetramic acid core have similarlybeen shown to have promising biological activity for a number ofimportant indications.⁹⁻¹¹

Synthesis of tricyclic chemical compounds containing the1,4-benzodiazepine and tetramic acid subunits has previously beendocumented.¹² Such tricyclic pyrrolobenzodiazepine derivatives have beensynthesized by the condensation reaction of enaminolactams with thecorresponding aldehydes.¹²⁻¹³ While some of these compounds producedinteresting biological activity,¹² the addition of the third ring ontothe benzodiazepine core would be expected to produce effects divergentfrom those of classical bicyclic fused benzodiazepines which mimicβ-turn peptide features.⁸

To the best of our knowledge there have been no reports on the synthesisof bicyclic systems resulting from the fusion of 1,4-diazepine and2,4-pyrrolidinedione subunits. This is interesting to note since thebicyclic ring system would be expected to possess lower CLogP valuesthan the tricylic system,¹⁴ making them potentially more suitable astherapeutics.

Herein, we describe the synthesis of a novel series of bicyclicpyrrolodiazepines comprised of the 1,4-diazepine and2,4-pyrrolidinedione subunits. This work enables the synthesis of a widevariety of diverse and novel drug-like compounds for potentialbiomedical applications. They may therefore be of great interest tomedicinal chemists looking to prepare molecules that access new chemicalspace for novel drug targets.

Results

The overall synthetic approach is based on previously reported synthesisof furobenzodiazepines and pyrrolobenzodiazepines by Matsuo andTanaka.¹³ Vinylogous urea 6 was synthesized in six steps fromcommercially available amino acid ester 1 as illustrated in Scheme 1.Reductive amination of amino acid ester 1 with benzaldehyde using NaBH₄as the reducing agent gave the corresponding secondary amine 2 inexcellent yield (98%). N-acylation of amine 2 with ethyl3-chloro-3-oxopropanoate in the presence of triethylamine (TEA) as basegave the intermediate diester. Interestingly, the intermediate acyclicamide could not be isolated as TEA was sufficient to promote theimmediate Dieckmann condensation to form 5-membered ring 3 in 60% yield.Hydrolysis-decarboxylation of keto-ester 3 using aqueous AcOH providedpyrrolidinedione 4 in excellent yield (82%). Boc-protected enaminolactam5 was obtained in 82% yield by treating 4 with tert-butyl(2-aminoethyl)carbamate in the presence of a catalytic amount of pTsOH.Finally, removal of the boc-protecting group afforded the desiredvinylogous urea 6 in excellent yield (92%) which could enable furtherwide structural diversification. This rapid and efficient six stepsynthetic route furnished vinylogous urea 6 in an overall 36% yield.

With the key vinylogous urea 6 in hand, we were able to synthesize thefinal pyrrolodiazepine-based compound library by diversification at twokey sites. Scheme 2 depicts the sequence for generation of the desiredlibrary of compounds Mannich-type condensation of 6 with variousaldehydes (R₁CHO) in the presence of AcOH proceeded smoothly to give thecorresponding bicyclic amines 7. These reaction conditions were found tobe compatible with various aldehydes including heteroaromatic andaliphatic aldehydes Amines 7a-c were found to degrade when purificationwas attempted using normal phase flash chromatography and were thereforeused without further purification. The crude amines (7a-c) wereimmediately subjected to chemoselective N-acylation reactions. Diverseacid chlorides (R₂COCl) including aliphatic, heterocyclic, and aromaticwere used to provide the final compounds 8a-8y. The sulfonamide couldalso be formed by using methanesulfonyl chloride and TEA (8j).

TABLE 1 Synthesis of bicyclic pyrrolodiazepines 8a-y

8a

8b

8c

8d

8e

8f

8g

8h

8i

8j

8k

8l

8m

8n:

8o

8p

8q

8r

8s

8t

8u

8v

8w

8x

8y

A subset of the compound library was submitted to the National CancerInstitute (NCI) for screening in their NCI60 panel for anti-canceractivity.¹⁵⁻¹⁷ Assay details are provided in the supplemental. Notably,compound 8f was found to reduce cell growth of A549 non-small cell lungcancer cells by 32% at a concentration of 10 μM. Compounds 8c and 8meach reduced of A549 cell growth by 24% at 10 μM. While the potency isless than optimal, we anticipate that the general synthetic route willfacilitate further optimization and exploration of these polycyclicscaffolds.

DISCUSSION

The diazepine and tetramic acid rings have proven to be privilegedstructures in biology and medicinal chemistry; however, there areremarkably few reports of heterocyclic scaffolds containing these tworing systems fused together. Here we report an efficient synthesis of aseries of novel fused bicyclic heterocycles incorporating these motifs.The synthesis allows for extensive diversification of substituents onthe unique 5,6-fused pyrrolodiazepinone ring system. We havedemonstrated that this chemistry is wide in scope and can allow accessto a large number of structurally diverse analogs. Yields for the finaltwo-step sequence which allows wide diversification were highly variableand ranged from low (15% for 8d) to good (68% for 8k). There was noobvious pattern which explained the wide-ranging yields. However, it wasobserved that many of the final acylation reactions produced a largenumber of impurities (as assessed by LC/MS). As the intermediate freeamines (7a-c) showed degradation to normal phase flash chromatography,it is possible that some of the amine degraded before the finalacylation reaction, resulting in lower yields. Also, acylation ofcompounds 7a-c is likely not entirely chemoselective. For instance,besides the desired path to final compounds 8, the intermediate 7a-c hasanother amine and an enamine-like motif, both of which could contributeto impurities.

New potential drug targets are constantly being uncovered in the searchfor ways to treat existing diseases. In particular, there is increasinginterest in developing small molecules that can modulateprotein-protein¹⁸⁻¹⁹ and protein-DNA/RNA²⁰⁻²¹ interactions. However,reliance on existing molecular space to pursue these challenging targetshas led to frustratingly little success in the development of drugsagainst new target classes. One reason for this is that current compoundlibraries that are used for high-throughput screening and which definethe chemotypes pursued in subsequent lead optimization cover very littlechemical space.²² In addition, they often over-represent scaffolds withhighly aromatic and flat structures which may be unsuitable fortargeting new types of drug targets.¹ Many of the new fused heterocycleswe describe in this report have very high fractions of sp^(a) (Fsp³)character. For example, compound 8e has very favorable physiochemicalproperties, with a Fsp³=0.56 and a CLogP=3.1.¹⁴ Future efforts thatreplace the 7-benzyl group with other less hydrophobic groups wouldenable the generation of even more diverse structures with lower CLogPvalues and potentially higher Fsp³ and solubility. Given the uniquenature of this fused scaffold, the privileged structural character fromwhich it was derived, and its highly 3-dimensional architecture, thismay be a useful chemotype for many current and future drug discoveryefforts.

To demonstrate the utility of this drug-like scaffold, several librarymembers were screened in the NCI-60 cell line panel to identifyanti-cancer activity.¹⁵⁻¹⁷ Four compounds were chosen that represented adiverse spectrum of structures: 8e, 8f, 8i, and 8m. These compounds hadboth aryl and alkyl groups at C-5, and at N-5, they had small cycloalkyland heteroaryl groups, thereby providing reasonable chemical spacecoverage from this set of available compounds. Notably, compound 8fshowed significant cytotoxicity against the non-small cell lung cancerline A549. Cell proliferation was reduced 32% at a single concentrationof 10 μM. In addition, compounds 8c and 8m also showed substantial cellgrowth inhibition as each reduced viability of A549 cells by 24% at 10μM. Compound 8e had essentially no effect on the proliferation of A549cells (<5% growth reduction). These results are especially significantsince these three compounds are all closely structurally-related membersof this drug-like bicylic system. This strongly suggests that thesecompounds share a common molecular target that may be exploitable forfuture development as a new anti-cancer strategy.

In conclusion, we have described the synthesis of a library of novelfused heterocyclic compounds. We expect that the synthetic routes wehave described will allow the generation of many new diverse moleculesfrom these potentially bioactive chemotypes. These molecules will enablechemical biology and drug discovery researchers to expand into newchemical space and explore new avenues for both basic and translationalresearch.

GENERAL EXPERIMENTAL

All chemical reagents were obtained from commercial suppliers and usedwithout further purification unless otherwise stated. Anhydrous solventswere purchased from Sigma-Aldrich, and dried over 3 Å molecular sieveswhen necessary. DCM and THF were purified by passage through a bed ofactivated alumina. Normal phase flash column chromatography wasperformed using Biotage KP-Sil 50 μm silica gel columns and ACS gradesolvents on a Biotage Isolera flash purification system. Analytical thinlayer chromatography (TLC) was performed on EM Reagent 0.25 mm silicagel 60 F₂₅₄ plates and visualized by UV light. Liquidchromatography/mass spectrometry (LCMS) was performed on a WatersAcquity-H UPLC system with a 2.1 mm×50 mm, 1.7 μm, reversed phase BEHC₁₈ column and LCMS grade solvents. A gradient elution from 95%water+0.1% formic acid/5% acetonitrile+0.1% formic acid to 95%acetonitrile+0.1% formic acid/5% water+0.1% formic acid over 2 min plusa further minute continuing this mixture at a flow rate of 0.85 mL/minwas used as the eluent. Total ion current traces were obtained forelectrospray positive and negative ionization (ESI+/ESI−). Proton (¹H),and carbon (¹³C) NMR spectra were recorded on a Bruker Avance III w/direct cryoprobe spectrometer. Chemical shifts were reported in ppm (δ)and were referenced using residual non-deuterated solvent as an internalstandard (CDCl₃ at 7.26 ppm for ¹H-NMR and 77.16 for ¹³C-NMR). Thechemical shifts for ¹H NMR and ¹³C NMR are reported to the seconddecimal place. Proton coupling constants are expressed in hertz (Hz).The following abbreviations were used to denote spin multiplicity forproton NMR: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,brs=broad singlet, dd=doublet of doublets, dt=doublet of triplets,quin=quintet, tt=triplet of triplets. In some cases, overlapping signalsoccurred in the ¹³C NMR spectra.

Procedures and Analytical Data for the Synthesis of Compounds 2-8

Methyl 2-(benzylamino)-2-methylpropanoate (2)

To a solution of methyl 2-amino-2-methylpropanoate hydrochloride 1 (503mg, 3.27 mmol) in THF (5 ml) was added MgSO₄ (300 mg, 3.27 mmol), TEA(0.456 ml, 3.27 mmol), and benzaldehyde (0.33 ml, 3.27 mmol). Thesuspension was stirred at room temperature overnight. The reactionsuspension was filtered, washed with THF, and the filtrate concentratedin vacuo. The residue obtained was taken up in MeOH (10 ml) and cooledto 0° C. NaBH₄ (149 mg, 3.93 mmol) was added in several portions over aperiod of 10 minutes. After the addition was complete, the reaction waswarmed to room temperature and stirred for 1 h. Water was added and theproduct was extracted with EtOAc. The organic layer was combined, washedwith brine, dried over anhydrous Na₂SO₄, filtered, and concentrated invacuo to afford compound 2 (620 mg, 98%) which was used in the next stepwithout further purification. ¹H NMR (500 MHz, CDCl₃) δ 1.34-1.41 (m,6H), 3.62 (br. s., 2H), 3.74 (br. s., 3H), 4.69 (br. s., 1H), 7.32-7.39(m, 5H).

Ethyl 1-benzyl-5,5-dimethyl-2,4-dioxopyrrolidine-3-carboxylate (3)

A solution of methyl 2-(benzylamino)-2-methylpropanoate 2 (3.4 g, 16.4mmol) in DCM (60 ml) was cooled to 0° C. and treated with TEA (5.03 ml,36.1 mmol). To this was added a solution of ethyl3-chloro-3-oxopropanoate (2.1 ml, 16.4 mmol) in 30 mL DCM dropwise. Thereaction was stirred at room temperature for 6 h. Water was added andthe layers were separated. The organic layer was washed with saturatedaqueous NaHCO₃, 1N HCl and brine, then dried over anhydrous Na₂SO₄,filtered and concentrated in vacuo to give an oily residue (mixture ofuncyclized intermediate and cyclized product). The residue was dissolvedin MeOH (6 mL) and treated with sodium methoxide (1.26 g, 23.34 mmol).The resulting mixture was heated at 60° C. for 1 h. The reaction wasthen cooled to room temperature and quenched with water. DCM was addedand the layers separated. The organic layer was washed with 1N HCl andwater, then dried over anhydrous Na₂SO₄ and evaporated to yield compound3 as an off-white solid (1.6 g, 60%). MS (ESI): mass calcd. forC₁₆H₁₉NO₄, 289.14; m/z found, 290.14 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ1.27 (s, 6H), 1.42 (t, J=7.02 Hz, 3H), 4.42 (d, J=7.32 Hz, 2H), 4.56 (s,2H), 7.23 (br. s., 1H), 7.29-7.35 (m, 4H).\

1-Benzyl-5,5-dimethylpyrrolidine-2,4-dione (4)

A mixture of ethyl1-benzyl-5,5-dimethyl-2,4-dioxopyrrolidine-3-carboxylate 3 (0.52 g, 1.8mmol), water (10 ml) and acetic acid (1 ml) was heated at 60° C. for 1h. The reaction was cooled to room temperatue and extracted with DCM.The organic layer was washed with water, then dried over anhydrousNa2SO4, filtered and concentrated in vacuo to afford compound 4 as apale-yellow solid (320 mg, 82%). MS (ESI): mass calcd. for C₁₃H₁₅NO₂,217.11; m/z found, 218.13 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ 1.22 (s,6H), 3.16 (s, 2H), 4.61 (s, 2H), 7.26-7.40 (m, 5H).

tert-Butyl(2-((1-benzyl-2,2-dimethyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)amino)ethyl)carbamate(5)

To a solution of 1-benzyl-5,5-dimethylpyrrolidine-2,4-dione 4 (500 mg,2.301 mmol) and tert-butyl (2-aminoethyl)carbamate (369 mg, 2.301 mmol)in benzene (12 ml) was added 4-methylbenzenesulfonic acid hydrate (43.8mg, 0.230 mmol). The resulting solution was heated to 80° C. and stirredat that temperature for 1 h. The solvents were removed in vacuo and theresulting residue was purified by flash chromatography on silica gel(0-10% MeOH in DCM) to afford compound 5 as a light pink solid (686 mg,82%). MS (ESI): mass calcd. for C₂₀H₂₉N₃O₃, 359.22; m/z found, 360.28[M+H]+; ¹H NMR (500 MHz, CDCl₃) δ 1.15 (s, 6H), 1.41 (s, 9H), 3.11-3.17(m, 2H), 3.37-3.44 (m, 2H), 4.54 (s, 2H), 4.66 (s, 1H), 4.90 (br. s.,1H), 5.40 (br. s., 1H), 7.17-7.23 (m, 2H), 7.28-7.32 (m, 3H).

4-((2-Aminoethyl)amino)-1-benzyl-5,5-dimethyl-1H-pyrrol-2(5H),-one (6)

Trifluoroacetic acid (1 ml) was added to a solution of tert-butyl(24(1-benzyl-2,2-dimethyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)amino)ethyl)carbamate5 (0.45 g, 1.25 mmol) in DCM (6 ml). The resulting solution was stirredat room temperature for 1 h. The solvents were evaporated under reducedpressure and the resulting residue was dried under high vacuum to yieldvinylogous urea 6 (300 mg, 92%) which was taken to the next step withoutfurther purification. MS (ESI): mass calcd. for C₁₅H_(2i)N₃O, 259.17;m/z found, 260.20 [M+H]+; ¹H NMR (500 MHz, CDCl₃) δ 1.22 (s, 6H), 3.16(s, 2H), 4.62 (s, 2H), 7.21 (s, 1H), 7.30-7.34 (m, 5H).

General Procedure a for the Synthesis of Bicyclic Amines 7a and 7b

To a solution of4-((2-aminoethyl)amino)-1-benzyl-5,5-dimethyl-1H-pyrrol-2(5H),-one 6(1.0 equiv) in ethanol (0.1 M) was added the respective aldehyde (1.1equiv). Acetic acid (1.1 equiv) was added and the resulting clearsolution was stirred at room temperature for 16 h. The mixture wasconcentrated in vacuo to give the respective bicylic amines (7a-7d)which were subsequently used without any purification.

7-benzyl-5-isopropyl-8,8-dimethyl-4-(methylsulfonyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8j)

Prepared according to a slightly modified version of General procedure Busing7-benzyl-5-isopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7a. Instead of using an acid chloride, 1 eq. of methanesulfonyl chloridewas used to afford7-benzyl-5-isopropyl-8,8-dimethyl-4-(methylsulfonyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8j (66%). MS (ESI): mass calcd. forC₂₀H₂₉N₃O₃S, 391.19; m/z found, 392.31 [M+H]+; ¹H NMR (500 MHz, CDCl₃) δ1.05 (d, J=6.71 Hz, 3H) 1.10 (s, 2H) 1.12 (s, 5H) 1.18 (s, 4H) 1.99-2.09(m, 1H) 2.85 (s, 3H) 3.29-3.38 (m, 1H) 3.48-3.58 (m, 3H) 3.96-4.05 (m,3H) 4.38-4.46 (m, 1H) 4.46-4.51 (m, 1H) 4.67-4.73 (m, 1H) 7.21-7.25 (m,3H) 7.29 (d, J=4.27 Hz, 5H); ¹³C NMR (126 MHz, CDCl₃) δ 19.90, 20.33,24.69, 24.94, 34.02, 39.70, 42.48, 45.92, 46.44, 58.47, 61.23, 104.26,127.18, 127.73, 128.58, 139.87, 164.13, 170.49.

7-benzyl-5-isopropyl-8,8-dimethyl-4-(thiophene-2-carbonyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8k)

Prepared according to General procedure B using7-benzyl-5-isopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7a to afford7-benzyl-5-isopropyl-8,8-dimethyl-4-(2-phenylacetyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H),-one 8k (68%). MS (ESI): mass calcd. forC₂₄H₂₉N₃O₂S, 423.20; m/z found, 424.32 [M+H]+; ¹H NMR (500 MHz, CDCl₃) δppm 0.92 (d, J=6.10 Hz, 5H) 1.01 (d, J=6.10 Hz, 5H) 1.15 (s, 6H) 1.21(s, 3H) 2.16 (d, J=8.24 Hz, 1H) 3.32 (t, J=12.51 Hz, 4H) 3.57 (br. s.,2H) 4.10 (br. s., 1H) 4.45 (d, J=15.56 Hz, 1H) 4.71 (d, J=16.17 Hz, 2H)4.74-4.79 (m, 1H) 5.07 (d, J=9.77 Hz, 1H) 7.09 (br. s., 1H) 7.20-7.25(m, 1H) 7.28-7.33 (m, 4H) 7.45 (d, J=5.19 Hz, 2H); ¹³C NMR (126 MHz,CDCl₃) δ 20.11, 20.16, 24.89, 24.97, 33.36, 42.44, 42.70, 46.90, 60.93,61.50, 103.43, 127.11, 127.28, 127.71, 128.52, 128.91, 129.25, 137.81,140.03, 164.63, 164.90, 170.47.

7-benzyl-5-isopropyl-4-(4-methoxybenzoyl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8l)

Prepared according to General procedure B using7-benzyl-5-isopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7a to afford7-benzyl-5-isopropyl-4-(4-methoxybenzoyl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8l (42%). MS (ESI): mass calcd. for C₂₇H₃₃N₃O₃,447.25; m/z found, 448.39 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 0.87 (d,J=6.26 Hz, 3H) 0.92 (d, J=6.65 Hz, 3H) 1.16 (s, 3H) 1.22 (s, 3H)2.03-2.16 (m, 1H) 3.21-3.30 (m, 1H) 3.31-3.40 (m, 1H) 3.56-3.65 (m, 1H)3.84 (s, 3H) 4.07-4.13 (m, 1H) 4.45-4.49 (m, 0H) 4.52 (s, 1H) 4.61 (s,1H) 4.65 (s, 1H) 4.74-4.83 (m, 1H) 6.92 (d, J=8.61 Hz, 2H) 7.23-7.26 (m,1H) 7.29-7.35 (m, 4H); ¹³C NMR (126 MHz, CDCl₃) δ 19.94, 20.09, 25.00,25.09, 33.48, 42.22, 42.45, 47.08, 55.41, 61.19, 61.30, 103.83, 113.95,127.11, 127.79, 128.50, 128.89, 140.29, 160.80, 164.47, 170.21, 171.92.

7-benzyl-4-(cyclobutanecarbonyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8m)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b (61 mg, 0.15 mmol) to afford7-benzyl-4-(cyclobutanecarbonyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8m (32 mg, 41%). MS (ESI): mass calcd. for C₂₅H₂₉N₃O₃, 419.22; m/zfound, 420.34 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ 1.18 (s, 3H) 1.21 (s,3H) 1.78-1.91 (m, 1H) 1.95-2.09 (m, 1H) 2.23-2.41 (m, 3H) 2.47-2.61 (m,1H) 2.87 (ddd, J=14.42, 10.60, 1.53 Hz, 1H) 3.31-3.39 (m, 1H) 3.41-3.48(m, 1H) 3.65 (t, J=8.70 Hz, 1H) 4.24 (d, J=2.75 Hz, 1H) 4.28-4.36 (m,1H) 4.46-4.55 (m, 1H) 4.59-4.66 (m, 1H) 6.02 (s, 1H) 6.05 (d, J=3.05 Hz,1H) 6.29 (dd, J=3.05, 1.83 Hz, 1H) 7.21-7.26 (m, 1H) 7.30 (d, J=4.58 Hz,4H) 7.36 (d, J=0.92 Hz, 1H); 13C NMR (126 MHz, CDCl₃) δ 17.86, 24.91,25.08, 25.15, 25.55, 37.45, 41.72, 42.27, 46.86, 51.66, 61.78, 97.82,108.54, 110.14, 127.02, 127.62, 128.42, 139.73, 142.63, 151.83, 163.20,169.90, 173.78.

4-(benzo[d][1,3]dioxole-5-carbonyl)-7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8n)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford4-(benzo[d][1,3]dioxole-5-carbonyl)-7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8n (25%). MS (ESI): mass calcd. for C₂₈H₂₇N₃O₅, 485.20; m/z found,486.29 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.20 (s, 5H) 1.22 (s, 4H)3.05 (br. s., 1H) 3.54-3.57 (m, 2H) 4.19 (br. s., 1H) 4.44 (d, J=15.56Hz, 2H) 4.60 (d, J=15.87 Hz, 1H) 5.99-6.03 (m, 2H) 6.06 (d, J=1.83 Hz,1H) 6.22 (s, 1H) 6.30 (dd, J=3.36, 1.83 Hz, 1H) 6.85 (d, J=7.93 Hz, 1H)7.17-7.24 (m, 3H) 7.29 (s, 4H) 7.42 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ24.92, 25.21, 29.84, 42.38, 42.66, 46.89, 54.54, 61.97, 97.37, 101.53,108.52, 109.03, 110.35, 121.98, 127.13, 127.74, 128.53, 129.13, 139.93,142.89, 147.79, 149.20, 152.14, 163.76, 169.74, 170.50.

7-benzyl-4-(4-fluorobenzoyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8o)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford47-benzyl-4-(4-fluorobenzoyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8o (32%). MS (ESI): mass calcd. for C₂₇H₂₆FN₃O₃, 459.20; m/z found,460.28 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.20 (s, 3H) 1.25 (s, 3H)3.01-3.13 (m, 1H) 3.53-3.59 (m, 2H) 4.20 (br. s., 1H) 4.40 (d, J=15.87Hz, 1H) 4.48 (d, J=15.26 Hz, 1H) 4.64 (d, J=15.87 Hz, 1H) 6.05 (d,J=3.36 Hz, 1H) 6.13 (s, 1H) 6.31 (dd, J=3.05, 1.83 Hz, 1H) 7.12-7.16 (m,2H) 7.23 (d, J=4.58 Hz, 2H) 7.29 (br. s., 3H) 7.42 (s, 1H) 7.69 (d,J=5.49 Hz, 1H) 8.08-8.13 (m, 1H); ¹³C NMR (126 MHz, CDCl₃) δ ppm 25.12,25.44, 42.49, 42.60, 47.13, 54.53, 61.98, 109.14, 110.45, 115.74,115.84, 115.92, 116.01, 127.24, 127.87, 128.59, 129.78, 129.84, 132.96,139.86, 143.01, 151.84, 163.42, 169.57.

7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-(2-phenylacetyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8p)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-(2-phenylacetyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8p (37%). MS (ESI): mass calcd. for C₂₈H₂₉N₃O₃, 455.22; m/z found,456.29 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.05 (s, 3H) 1.20 (s, 3H)2.87-2.98 (m, 1H) 3.32-3.41 (m, 1H) 3.43 (br. s., 1H) 3.99-4.03 (m, 1H)4.04 (d, J=3.05 Hz, 1H) 4.06-4.12 (m, 1H) 4.35 (d, J=14.34 Hz, 1H) 4.47(s, 1H) 4.56 (s, 1H) 5.96-6.02 (m, 1H) 6.26-6.30 (m, 2H) 7.28-7.31 (m,7H) 7.34-7.37 (m, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 24.90, 24.96, 41.00,42.08, 42.34, 46.62, 53.03, 61.95, 96.54, 108.67, 110.35, 126.75,127.14, 127.76, 128.48, 128.63, 129.37, 135.03, 139.79, 142.72, 151.92,163.31, 169.86, 170.41.

7-benzyl-5-(furan-2-yl)-4-isonicotinoyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8q)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford7-benzyl-5-(furan-2-yl)-4-isonicotinoyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8q (37%). MS (ESI): mass calcd. for C₂₆H₂₆N₄O₃,442.20; m/z found, 443.27 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.21 (s,3H) 1.26 (s, 3H) 3.06-3.14 (m, 1H) 3.50-3.64 (m, 2H) 4.22-4.29 (m, 1H)4.42 (s, 1H) 4.47-4.53 (m, 1H) 4.62-4.68 (m, 1H) 6.00 (s, 1H) 6.05-6.08(m, 1H) 6.30-6.34 (m, 1H) 7.24-7.34 (m, 6H), 7.44 (d, J=1.83 Hz, 1H)7.53-7.57 (m, 2H) 8.75 (d, J=5.80 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δppm 14.14, 22.68, 24.99, 25.35, 31.61, 42.26, 42.36, 46.89, 54.18,61.89, 97.44, 109.21, 110.39, 121.47, 127.13, 127.75, 128.47, 139.64,143.02, 150.39, 151.15, 152.50.

7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-nicotinoyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8r)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-nicotinoyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8r (50%). MS (ESI): mass calcd. for C₂₆H₂₆N₄O₃,442.20; m/z found, 443.28 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.22 (s,3H), 1.24 (s, 3H), 3.07-3.17 (m, 1H), 3.57-3.63 (m, 2H), 4.22-4.26 (m,1H), 4.42-4.46 (m, 1H), 4.48-4.53 (m, 1H), 4.58-4.64 (m, 1H), 6.03-6.09(m, 2H), 6.30-6.34 (m, 1H), 7.20-7.30 (m, 3H), 7.38-7.45 (m, 2H),8.01-8.06 (m, 1H), 8.68-8.72 (m, 1H), 8.91-8.96 (m, 1H); ¹³C NMR (126MHz, CDCl₃) δ 24.82, 25.17, 42.36, 42.56, 46.72, 54.53, 62.15, 96.20,109.28, 110.46, 123.56, 127.18, 127.65, 128.55, 131.70, 135.12, 139.67,143.09, 148.35, 150.82, 151.51, 164.10, 168.37, 169.64.

7-benzyl-4-(cyclopentanecarbonyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8s)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford7-benzyl-4-(cyclopentanecarbonyl)-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8s (39%). MS (ESI): mass calcd. for C₂₆H₃₁N₃O₃, 433.24; m/z found,434.33 [M+H]+; ¹H NMR (500 MHz, CDCl₃) δ ppm 1.21-1.22 (m, 6H),1.62-1.66 (m, 4H), 1.70-1.82 (m, 4H), 1.94-2.01 (m, 1H), 2.02-2.08 (m,1H), 2.86-2.96 (m, 1H), 3.25-3.40 (m, 2H), 3.43-3.50 (m, 1H), 4.11-4.15(m, 1H), 4.31-4.38 (m, 1H), 4.48-4.54 (m, 1H), 4.62-4.68 (m, 1H),6.07-6.09 (m, 1H), 6.30-6.32 (m, 2H), 7.24-7.32 (m, 5H), 7.38 (s, 1H);¹³C NMR (126 MHz, CDCl₃) δ 25.10, 25.35, 26.26, 26.32, 30.48, 30.61,41.52, 42.08, 42.50, 47.14, 52.20, 61.97, 98.07, 108.69, 110.32, 127.20,127.83, 128.59, 139.90, 142.80, 152.24, 163.35, 170.06, 175.42.

4-benzoyl-7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8t)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b (61 mg, 0.18 mmol) to afford4-benzoyl-7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8t (39 mg, 48%). MS (ESI): mass calcd. for C₂₇H₂₇N₃O₃, 441.21; m/zfound, 442.27 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ 1.15-1.18 (m, 3H) 1.21(s, 3H) 3.04 (ddd, J=14.50, 7.93, 4.12 Hz, 1H) 3.46-3.56 (m, 2H) 4.39(d, J=15.87 Hz, 1H) 4.44 (br. s., 1H) 4.46-4.52 (m, 1H) 4.63 (d, J=15.87Hz, 1H) 6.04 (d, J=3.05 Hz, 1H) 6.13 (s, 1H) 6.30 (dd, J=3.05, 1.83 Hz,1H) 7.20-7.26 (m, 1H) 7.27-7.32 (m, 4H) 7.40-7.46 (m, 5H) 7.58-7.68 (m,2H); 13C NMR (126 MHz, CDCl₃) δ 24.89, 25.21, 42.23, 46.91, 54.28,61.78, 97.47, 108.83, 110.19, 127.00, 127.15, 127.63, 128.38, 130.02,125.33, 139.78, 142.75, 151.86, 163.29, 169.46, 170.95.

7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-(thiophene-2-carbonyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8u)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b (12 mg, 0.035 mmol) to afford7-benzyl-5-(furan-2-yl)-8,8-dimethyl-4-(thiophene-2-carbonyl)-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8u (3 mg, 19%). MS (ESI): mass calcd. for C₂₅H₂₅N₃O₃S, 447.16; m/zfound, 448.27 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ 1.19-1.24 (m, 6H)3.07-3.18 (m, 1H) 3.46-3.52 (m, 2H) 3.54-3.61 (m, 2H) 3.66-3.72 (m, 1H)4.23 (br. s., 1H) 4.36-4.41 (m, 1H) 4.46 (d, J=16.04 Hz, 1H) 4.62 (s,1H) 6.14 (br. s., 1H) 6.34 (dd, J=3.13, 1.96 Hz, 1H) 6.68 (br. s., 1H)7.06-7.12 (m, 1H) 7.20-7.26 (m, 2H) 7.28-7.32 (m, 3H) 7.44 (s, 1H) 7.49(d, J=5.09 Hz, 1H) 7.79 (br. s., 1H); ¹³C NMR (126 MHz, CDCl₃) δ 25.01,25.38, 42.45, 43.37, 46.95, 54.48, 62.00, 97.76, 109.11, 110.45, 127.18,127.32, 127.81, 128.57, 129.49, 129.89, 137.26, 139.87, 142.99, 152.06,163.35, 164.11, 169.78.

7-benzyl-5-(furan-2-yl)-4-(4-methoxybenzoyl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8v)

Prepared according to General procedure B using7-benzyl-5-(furan-2-yl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H),-one7b to afford7-benzyl-5-(furan-2-yl)-4-(4-methoxybenzoyl)-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8v (25%). MS (ESI): mass calcd. for C₂₈H₂₉N₃O4,471.22; m/z found, 472.35 [M+H]+; ¹H NMR (500 MHz, CDCl₃) δ 1.20 (s, 3H)1.21-1.24 (m, 3H) 2.98-3.11 (m, 1H) 3.53-3.60 (m, 2H) 3.84 (s, 3H) 4.24(br. s., 1H) 4.42 (d, J=15.65 Hz, 1H) 4.48 (d, J=15.26 Hz, 1H) 4.64 (d,J=15.65 Hz, 2H) 6.06 (d, J=3.13 Hz, 1H) 6.26 (s, 1H) 6.31 (dd, J=3.13,1.96 Hz, 1H) 6.95 (d, J=8.61 Hz, 2H) 7.21-7.26 (m, 1H) 7.29 (d, J=4.70Hz, 4H) 7.42 (s, 1H) 7.66 (d, J=9.00 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃) δ25.08, 25.41, 42.44, 42.65, 47.16, 54.59, 55.47, 61.91, 98.10, 108.97,110.37, 114.08, 127.16, 127.59, 127.85, 128.55, 129.49, 139.98, 142.86,152.26, 161.12, 163.37, 169.70, 170.98.

4-acetyl-7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8w)

Prepared according to General procedure B using7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one7c to afford74-acetyl-7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8w (19%). MS (ESI): mass calcd. for C₂₁H₂₇N₃O₂,353.21; m/z found, 354.34 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm0.24-0.31 (m, 1H) 0.43-0.51 (m, 1H) 0.63-0.69 (m, 1H) 0.73 (dd, J=9.92,5.04 Hz, 1H) 1.14 (s, 3H) 1.15 (s, 3H) 2.20 (s, 3H) 3.31 (d, J=12.21 Hz,3H) 3.45-3.53 (m, 5H) 4.02 (br. s., 3H) 4.49-4.64 (m, 12H) 7.24 (dd,J=8.85, 4.27 Hz, 2H) 7.28-7.33 (m, 5H); ¹³C NMR (126 MHz, CDCl₃) δ 2.21,5.23, 15.06, 22.18, 24.98, 25.15, 41.61, 42.48, 46.99, 57.88, 61.80,100.23, 127.15, 127.69, 128.55, 139.74, 163.20, 169.87, 170.66.

7-benzyl-4-(cyclobutanecarbonyl)-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8x)

Prepared according to General procedure B using7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one7c to afford7-benzyl-4-(cyclobutanecarbonyl)-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one8x (10%). MS (ESI): mass calcd. for C₂₄H₃₁N₃O₂, 393.24; m/z found,394.37 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm 0.21-0.27 (m, 1H) 0.39-0.48(m, 1H) 0.65 (d, J=2.74 Hz, 2H) 0.86 (d, J=19.96 Hz, 1H) 1.01-1.07 (m,1H) 1.11 (s, 3H) 1.14 (s, 2H) 1.73-1.84 (m, 1H) 1.95-2.07 (m, 1H)2.13-2.28 (m, 3H) 2.33-2.43 (m, 1H) 3.23-3.33 (m, 1H) 3.39 (s, 1H)3.45-3.52 (m, 2H) 3.95-3.99 (m, 1H) 4.45-4.56 (m, 3H) 7.20-7.25 (m, 1H)7.28-7.30 (m, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 2.05, 5.22, 14.70, 17.96,25.00, 25.07, 25.19, 25.72, 37.64, 41.57, 42.45, 47.04, 56.11, 61.66,101.23, 127.12, 127.70, 128.55, 139.92, 163.12, 170.50, 173.70.

4-benzoyl-7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one(8y)

Prepared according to General procedure B using7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4]diazepin-6(1H)-one7c to afford4-benzoyl-7-benzyl-5-cyclopropyl-8,8-dimethyl-2,3,4,5,7,8-hexahydropyrrolo[3,4-e][1,4] diazepin-6(1H)-one 8y (19%). MS (ESI): mass calcd. for C₂₆H₂₉N₃O₂,415.23; m/z found, 416.35 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) δ ppm0.06-0.14 (m, 1H) 0.37-0.44 (m, 1H) 0.50-0.60 (m, 1H) 0.89 (s, 2H)1.03-1.15 (m, 2H) 1.17 (s, 2H) 1.20 (s, 2H) 3.40-3.50 (m, 1H) 3.58-3.69(m, 2H) 4.05-4.13 (m, 1H) 4.45-4.61 (m, 2H) 4.74-4.83 (m, 1H) 7.22-7.26(m, 1H) 7.28-7.31 (m, 2H) 7.33-7.38 (m, 2H) 7.39-7.43 (m, 2H); ¹³C NMR(126 MHz, CDCl₃) δ 2.30, 5.10, 15.18, 24.95, 25.09, 41.86, 42.37, 47.06,58.88, 61.75, 100.28, 126.59, 127.07, 127.60, 128.48, 128.70, 129.86,136.01, 139.97, 163.99, 170.13, 170.82.

TABLE 2 Synthesized Molecules and Compounds Structure Molecule NameCompound ID

NUCC-0202791

NUCC-0202790

NUCC-0202789

NUCC-0202788

NUCC-0202787

NUCC-0202786

NUCC-0202785

NUCC-0202784

NUCC-0202783

NUCC-0202782

NUCC-0202781

NUCC-0202780

NUCC-0202779

NUCC-0202778

NUCC-0202777

NUCC-0202776

NUCC-0202775

NUCC-0202774

NUCC-0202773

NUCC-0202520

NUCC-0202519

NUCC-0202518

NUCC-0202517

NUCC-0202516

NUCC-0202515

NUCC-0202514

NUCC-0202513

NUCC-0202512

NUCC-0202511

NUCC-0202510

NUCC-0202509

NUCC-0202508

NUCC-0202507

NUCC-0202506

NUCC-0202505

NUCC-0202504

NUCC-0202503

NUCC-0202502

NUCC-0202501

NUCC-0202500

NUCC-0202499

NUCC-0202498

NUCC-0202497

NUCC-0202496

NUCC-0202495

NUCC-0202494

NUCC-0202493

NUCC-0077068

NUCC-0077067 8s

NUCC-0077066 8r

NUCC-0077065 8q

NUCC-0077064 8p

NUCC-0077063 8o

NUCC-0077062 8n

NUCC-0077061 8d

NUCC-0077060 8c

NUCC-0077059 8f

NUCC-0077058 8e

NUCC-0077057 8i

NUCC-0077056 8h

NUCC-0077055 8g

NUCC-0077054

NUCC-0077053

NUCC-0077052 8y

NUCC-0077051 8x

NUCC-0077050 8w

NUCC-0077049 8l

NUCC-0077048 8k

NUCC-0077047 8v

NUCC-0077046 8u

NUCC-0077045 8t

NUCC-0077044 8m

NUCC-0077043 8b

NUCC-0077042 8a

NUCC-0075249 8j

NUCC-0060873

NUCC-0060872

NUCC-0060871

NUCC-0060870

NUCC-0060869

NUCC-0060868

NUCC-0060867

NUCC-0060866

NUCC-0060865

NUCC-0060864

NUCC-0060863

NUCC-0060862

NUCC-0060861

NUCC-0060860

NUCC-0060859

NUCC-0060858

NUCC-0060857

NUCC-0060856

NUCC-0060855

NUCC-0060854

NUCC-0054368

NUCC-0054140

Assay Protocol for Anti-Cancer Activity

Anti-cancer activity screening of all compounds was performed by the NCIDevelopmental Therapeutics Program (DTP) through their NCI60 screeninginitiative. Assay details can be found at the websitedtp.cancer.gov/discovery_development/nci-60/methodology.htm. Informationfrom this website is reproduced here for convenience.

NCI-60 Screening Methodology/NCI 60 Cell One-Dose Screen

General Description

As of early 2007 all compounds submitted to the NCI 60 Cell screen aretested initially at a single high dose (10-5 M) in the full NCI 60 cellpanel. Only compounds which satisfy pre-determined threshold inhibitioncriteria in a minimum number of cell lines will progress to the full5-dose assay. The threshold inhibition criteria for progression to the5-dose screen was selected to efficiently capture compounds withanti-proliferative activity based on careful analysis of historical DTPscreening data. The threshold criteria may be updated as additional databecomes available.

Interpretation of One-Dose Data

The One-dose data will be reported as a mean graph of the percent growthof treated cells and will be similar in appearance to mean graphs fromthe 5-dose assay. The number reported for the One-dose assay is growthrelative to the no-drug control, and relative to the time zero number ofcells. This allows detection of both growth inhibition (values between 0and 100) and lethality (values less than 0). This is the same as for the5-dose assay, described below. For example, a value of 100 means nogrowth inhibition. A value of 40 would mean 60% growth inhibition. Avalue of 0 means no net growth over the course of the experiment. Avalue of −40 would mean 40% lethality. A value of −100 means all cellsare dead. Information from the One-dose mean graph is available forCOMPARE analysis.

NCI 60 Cell Five-Dose Screen

Compounds which exhibit significant growth inhibition in the One-DoseScreen are evaluated against the 60 cell panel at five concentrationlevels.

The human tumor cell lines of the cancer screening panel are grown inRPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells are inoculated into 96 wellmicrotiter plates in 100 μL at plating densities ranging from 5,000 to40,000 cells/well depending on the doubling time of individual celllines. After cell inoculation, the microtiter plates are incubated at37° C., 5% CO2, 95% air and 100% relative humidity for 24 h prior toaddition of experimental drugs.

After 24 h, two plates of each cell line are fixed in situ with TCA, torepresent a measurement of the cell population for each cell line at thetime of drug addition (Tz). Experimental drugs are solubilized indimethyl sulfoxide at 400-fold the desired final maximum testconcentration and stored frozen prior to use. At the time of drugaddition, an aliquot of frozen concentrate is thawed and diluted totwice the desired final maximum test concentration with complete mediumcontaining 50 μg/ml gentamicin. Additional four, 10-fold or ½ log serialdilutions are made to provide a total of five drug concentrations pluscontrol. Aliquots of 100 μl of these different drug dilutions are addedto the appropriate microtiter wells already containing 100 μl of medium,resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48 hat 37° C., 5% CO2, 95% air, and 100% relative humidity. For adherentcells, the assay is terminated by the addition of cold TCA. Cells arefixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA(final concentration, 10% TCA) and incubated for 60 minutes at 4° C. Thesupernatant is discarded, and the plates are washed five times with tapwater and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4%(w/v) in 1% acetic acid is added to each well, and plates are incubatedfor 10 minutes at room temperature. After staining, unbound dye isremoved by washing five times with 1% acetic acid and the plates are airdried. Bound stain is subsequently solubilized with 10 mM trizma base,and the absorbance is read on an automated plate reader at a wavelengthof 515 nm. For suspension cells, the methodology is the same except thatthe assay is terminated by fixing settled cells at the bottom of thewells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA).Using the seven absorbance measurements [time zero, (Tz), controlgrowth, (C), and test growth in the presence of drug at the fiveconcentration levels (Ti)], the percentage growth is calculated at eachof the drug concentrations levels. Percentage growth inhibition iscalculated as:

[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz

[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimentalagent. Growth inhibition of 50% (GI50) is calculated from[(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation. The drug concentrationresulting in total growth inhibition (TGI) is calculated from Ti=Tz. TheLC50 (concentration of drug resulting in a 50% reduction in the measuredprotein at the end of the drug treatment as compared to that at thebeginning) indicating a net loss of cells following treatment iscalculated from [(Ti−Tz)/Tz]×100=−50. Values are calculated for each ofthese three parameters if the level of activity is reached; however, ifthe effect is not reached or is exceeded, the value for that parameteris expressed as greater or less than the maximum or minimumconcentration tested.

TABLE 3 Growth Inhibition of NUCC-608455 on NCI60 Cell Line PanelPanel/Cell Line Growth Percent Leukemia CCRF-CEM 24.15 HL-60(TB) 9.63K-562 12.60 MOLT-4 28.58 RPMI-

226 23.43 SR 23.29 Non-Small Cell Lung Cancer A549/ATCC 22.02 EKVX 35.37HOP-

2 33.09 HOP-92 21.07 NCI-H226 6

.66 NCI-H23 20.65 NCI-H322M 44.61 NCI-H4

0 4.63 NCI-H522 21.79 Colon Cancer COLO 205 1

.77 HCC-2998 3.73 HCT-116 7.52 HCT-15 15.65 HT29 9.99 KM12 14.83 SW-62021.68 CNS Cancer SF-268 42.68 SF-295 −0.75 SF-539 −17.85 SNB-19 23.61SNB-75 1

.63 U251 24.64 Melanoma LOX

MVI 25.57 MALME-3M 69.89 M14 −13.5

MDA-MB-435 −5

.24 SK-MEL-2 16.91 SK-MEL-28 57.2

SK-MEL-5 7.88 UACC-257 77.61 UACC-62 21.25 Ovarian Cancer IGROV1 26.78OVCAR-3 −7.65 OVCAR-4 48.83 OVCAR-5 25.92 OVCAR-8 2

.13 NC

ADR-RES 82.92 SK-OV-3 22.71 Renal Cancer 786-0 21.45 ACHN 33.51 CAKI-142.97 RXF 393 −11.72 SN12C 35.91 TK-10 54.92 UO-31 52.54 Prostate CancerPC-3 29.67 DU-145 7.82 Breast Cancer MCF7 15.26 MDA-MB-231/ATCC 9.25 HS57

T 10.

7 BT-549 47.62 T-47D 35.10 MDA-MB-46

−1.86 Mean 23.48 Delta 79.72 Range 139.1

indicates data missing or illegible when filed

TABLE 4 Growth Inhibition of NUCC-60872 on NCI60 Cell Line PanelPanel/Cell Line Growth Percent Leukemia CCRF-CEM 20.66 HL-60(TB) 7.50K-562 16.57 MOLT-4 33.27 RPMI-

226 12.61 SR 16.70 Non-Small Cell Lung Cancer A549/ATCC 80.32 EKVX 61.53HOP-

2 61.59 HOP-92 37.54 NCI-H226 91.06 NCI-H23 50.21 NCI-H322M 73.36 NCI-H4

0 21.87 NCI-H522 18.58 Colon Cancer COLO 205 14.23 HCC-2998

0.81 HCT-116 24.91 HCT-15 93.83 HT29 25.22 KM12 19.79 SW-620 24.24 CNSCancer SF-268 42.99 SF-295 45.97 SF-539 20.14 SNB-19 39.

7 SNB-75 −3.29 U251 18.07 Melanoma LOX

MVI 37.52 MALME-3M 44.26 M14 21.48 MDA-MB-435 −67.83 SK-MEL-2 8.92SK-MEL-2

56.02 SK-MEL-5 27.50 UACC-257 36.37 UACC-62 32.33 Ovarian Cancer IGROV139.64 OVCAR-3 18.09 OVCAR-4 55.16 OVCAR-5 62.50 OVCAR-8 65.

4 NC

ADR-RES 101.74 SK-OV-3 55.42 Renal Cancer 786-0 80.09 ACHN 95.96 CAKI-171.91 RXF 393 27.79 SN12C 40.74 TK-10 87.08 UO-31 81.99 Prostate CancerPC-3 36.41 DU-145 78.9

Breast Cancer MCF7 16.41 MDA-MB-231/ATCC 63.32 HS 57BT 20.42 BT-54969.46 T-47D 26.72 MDA-MB-46

19.19 Mean 41.43 Delta 109.2

Range 169.57

indicates data missing or illegible when filed

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In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A compound or a salt thereof having a formula:

wherein: R¹ and R² are H or alkyl; R³ is H, alkyl, alkenyl, or benzyl;R⁴ is H, alkyl, benzyl, a carbocycle, a heterocycle, two fusedcarbocycles, two fused heterocycles, or a fused carbocycle andheterocycle, which carbocycle and heterocycle are saturated orunsaturated at one or more bonds, and R¹⁴ optionally is substituted atone or more positions with halo, alkyl, haloalkyl, alkyloxy, orcarboxyl; R^(4′) is H or alkyl; R⁵ is H, alkyl, aryl, or alkylaryl, andR⁵ optionally is substituted at one or more positions with halo, alkyl,haloalkyl, alkoxy, carboxy, or amino which optionally is substitutedwith alkyl, or R⁵ has a formula

 wherein R¹⁰ is H, alkyl, phenylamino, or benzyl, or R¹⁰ is acarbocycle, a heterocycle, two fused carbocycles, two fusedheterocycles, or a fused carbocycle and a fused heterocycle, whichcarbocycle and heterocycle are saturated or unsaturated at one or morebonds and R¹⁰ optionally is substituted at one or more positions withhalo, alkyl, haloalkyl, alkoxy, carboxy, or amino which optionally issubstituted with alkyl; or R¹⁰ has a formula

 wherein R¹³ is H or alkyl, R¹⁴ is H or alkyl, R¹⁵ is benzyl, oxybenzyl,or a carbocycle, a heterocycle, two fused carbocycles, two fusedheterocycles, or a fused carbocycle and heterocycle, which carbocycleand heterocycle are saturated or unsaturated at one or more bonds andR¹⁵ optionally are substituted at one or more positions with halo,alkyl, haloalkyl, alkoxy, carboxy, or amino which optionally issubstituted with alkyl; and R⁶ and R⁷ are H, or R⁶ and R⁷ together formphenyl fused to the 1.4-diazepine core.
 2. The compound of claim 1,wherein R¹ and R² are methyl.
 3. The compound of claim 1, wherein R³ ismethyl.
 4. The compound of claim 1, wherein R⁴ is selected from methyl,isopropyl, cyclopropyl, cyclobutyl, furanyl, pyridinyl, phenylfluorophenyl, chlorophenyl, carboxyphenyl, methoxyphenyl benzyl,thiophenyl chlorothiophenyl, tetrahydrofuranyl, thiazolyl,1,3-benzodioxolyl, 1,4-benzodioxanyl, and piperidinyl.
 5. The compoundof claim 1, wherein R⁵ has a formula

and R¹⁰ is methyl, tetrabutyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, phenyl, butyl, thiophenyl, 4-(N,N-diakylamino)phenyl,4-trifluoromethylphenyl, 4-methoxyphenyl, 4-fluorophenyl, phenylamino,1,3-benzodioxolyl, pyridinyl, amino, dialkylamino, or tetrahydropyranyl.6. The compound of claim 1, wherein R⁵ has a formula

R¹⁰ has a formula

R¹³ is H, R¹⁴ is H, and R¹⁵ is phenyl, oxybenzyl, tetrahydropyranyl, or1,3-benzodioxolyl.
 7. The compound of claim 1, wherein R⁴ is3,4-dichlorophenyl and R⁵ is oxobenzyl.
 8. A pharmaceutical compositioncomprising any effective amount of the compound of claim 1 and apharmaceutical carrier, excipient, or diluent.
 9. A method for treatinga disease or disorder in a subject in need thereof, the methodcomprising administering to the subject the pharmaceutical compositionof claim
 8. 10. The method of claim 9, wherein the disease or disorderis a cell proliferative disease or disorder.
 11. The method of claim 9,wherein the disease or disorder is cancer.
 12. A compound selected fromcompounds listed in Table 2 in the Specification that accompanies theseclaims.
 13. A pharmaceutical composition comprising any effective amountof the compound of claim 12 and a pharmaceutical carrier, excipient, ordiluent.
 14. A method for treating a disease or disorder in a subject inneed thereof, the method comprising administering to the subject thepharmaceutical composition of claim
 13. 15. The method of claim 14,wherein the disease or disorder is a cell proliferative disease ordisorder.
 16. A compound having a structure:


17. A compound having a structure:

wherein R¹ and R² are H or alkyl and R¹¹ and R¹² are H or an aminoprotecting group.
 18. The compound of claim 17, wherein R¹ and R² aremethyl and R¹¹ and R¹² are H.
 19. A compound having a structure:

wherein R¹ and R² are H or alkyl.
 20. The compound of claim 19, whereinR¹ and R² are methyl.